Phosphoprotein phosphatases regulate cellular metabolism together with protein kinases by modulating the level of phosphorylation and, hence, specific activity of regulatory proteins. A variety of multimeric phosphatases have been isolated and grouped into several classes, termed type 1 (ATP-Mg-dependent) and types 2A (e.g., eIF-2 phosphatase and smooth muscle phosphatase I), 2B (calcineurin) and 2C; they differ in their sensitivity to inhibitor proteins and divalent cations and in their substrate specificity. However, definitive characterization of the enzymes has been hampered by difficulties in maintaining their native state throughout biochemical purification procedures. To elucidate the relationship between different phosphoprotein phosphatases, highly purified enzymes from soil amoebae, turkey gizzards, bovine heart and brain, and rabbit skeletal muscle and reticulocytes, were tested for antigenic relatedness. Two heterologous antibody preparations were employed for this purpose -- one was made against an Acanthamoeba type 2A phosphatase and the other was made to bovine brain phosphatase type 2B (gift of Claude Klee). Specific subunit cross-reactivity was examined by protein blot ("Western") analysis. In addition, conditions were sought for purification of the type-2A phosphatase by immunoaffinity techniques. To this end, a modification of the dot immunobinding assay was developed and employed to screen for reagents to dissociate the enzyme gently from the immune complex without destroying enzyme activity. Immunoaffinity columns were prepared and tested for their ability to remove the enzyme from solution. Finally, immunotitrations were performed to determine the specific antibody titer and test for inhibition of enzyme activity.